Green synthesis of novel stable biogenic gold nanoparticles for breast cancer therapeutics via the induction of extrinsic and intrinsic pathways

Biosynthesis of gold nanoparticles (AuNPs) using algal polysaccharides is a simple, low-cost, and an eco-friendly approach. In the current study, different concentrations of Arthospira platensis exopolysaccharides (EPS) were used to synthetize AuNPs via the reduction of gold ions. The biologically synthesized AuNPs (AuNPs1, AuNPs2, AuNPs3) were prepared in 3 different forms through the utilization of three different ratios of EPS-reducing agents. AuNPs analysis confirmed the spherical shape of the EPS-coated AuNPs. Furthermore, AuNPs prepared by EPS and l-ascorbic acid (AuNPs3) showed more stability than the AuNPs colloidal solution that was prepared using only l-ascorbic acid. Analysis of the antimicrobial effects of AuNPs showed that E. coli was the most sensitive bacterial species for AuNPs3 and AuNPs1 with inhibition percentages of 88.92 and 83.13%, respectively. Also, safety assay results revealed that AuNPs3 was the safest biogenic AuNPs for the tested noncancerous cell line. The anticancer assays of the biogenic AuNPs1, AuNPs2, and AuNPs3 against MCF-7 cell line indicated that this cell line was the most sensitive cell line to all treatments and it showed inhibition percentages of 66.2%, 57.3%, and 70.2% to the three tested AuNPs, respectively. The AuNPs also showed abilities to arrest MCF-7 cells in the S phase (77.34%) and increased the cellular population in the sub G0 phase. Gene expression analysis showed that AuNPs3 down regulated Bcl2, Ikapα, and Survivn genes in MCF-7 treated-cells. Also, transmission electron microscopy (TEM) analysis of MCf-7 cells revealed that AuNPs 3 and AuNPs2 were localized in cell vacuoles, cytoplasm, and perinuclear region.


Characterization of AuNPs.
AuNPs stability using UV-Vis spectral analysis. The obtained data illustrated in Fig. 1a display the absorption peaks of the three different samples of gold nanoparticles (AuNPs1, AuNPs2, AuNPs3). The three spectra were normalized at the maximum Plasmon absorptions at 530.0, 540.0, and 550.0 nm. The aggregation state was detected visually by observing the change in solution color from red to blue or purple. Blue shift was observed here from 550 to 530 nm. Furthermore, the UV-Vis spectrum of AuNP3 that were prepared with polysaccharides and l-ascorbic acid was staple and didn't record any significant changes even after more than 3 months comparing with nanogold colloidal solution, which was prepared with only lascorbic acid as a reducing and stabilizing agent (Fig. 1b).
X-ray powdered diffraction (XRD). AuNPs were investigated using XRD to confirm their crystalline structures, where all of them expressed the most characteristic peaks of metallic gold. Four intensive Bragg reflections were observed in each case around 38.0996°, 44.3687°, 64.6765°, and 77.5471° corresponding to Miller indices (1 1 1), (2 0 0), (2 2 0), and (3 1 1). This confirmed the face-centered cubic crystalline symmetry of gold nanoparticles (JCPDS file no. 01-1174). The ratios between the intensities of (2 0 0) and (1 1 1) diffraction peaks were 0.31 in case of AuNPs1, 0.26 in case of AuNPs2, and 0.32 in case of AuNPs3 (Fig. 2a,b,c). All intensity ratios were lower than the conventional bulk intensity ratio of ~ 0.52. These findings confirmed that (1 1 1) is the preferential or predominant orientation, as confirmed previously by the following TEM studies. Nanoparticles sizes were estimated using XRD measurements (Table 1)

by applying Debye Scherrer equation.
Transmission electron microscopy (TEM). The morphology of the biogenic AuNPs was investigated using TEM scaning. The results revealed that spherical shape was the predominant shape in all the AuNPs preparations . AuNPs1 particle size that was estimated from TEM analysis ranged from 9.0 to 30.0 nm (Fig. 3). While, AuNPs2 showed a range of particle sizes between 8.0 and 35.0 nm, and AuNPs3 have particle sizes ranged from 6.0 to 40.0 nm (Fig. 3b).
High-resolution TEM imaging indicated that AuNPs1 showed only a single nano-crystal structure that appeared with clear lattice fringes with spacing of 0.234 nm (Fig. 3c). This result confirmed that nanogold crystals grow preferentially on the (1 1 1) plane (Fig. 2). This was confirmed by the d-spacing of (1 1 1) plane (provided from XRD measurement), which is equal to 0.2364 nm and the calculated interplanar distance of Au (1 1 1) plane. www.nature.com/scientificreports/ FTIR analysis. FTIR analysis of algal polysaccharides. FTIR analysis was used to analyze the functional groups of the extracted polysaccharides and to identify which biomolecules are responsible for the reduction and capping of the prepared biogenic gold nanoparticles. The FTRI spectrum of the extracted polysaccharides showed peaks at 1464.0, 1699.0, 3070.0, and 3466.0 cm −1 . In addition, multiple peaks were observed at 665.0, 846.0, and 1008.0-1190.0 cm −1 . A broad intense band at 3466.0 cm −1 was also observed, which could be assigned to OH groups of algal polysaccharides or a secondary amide group (Fig. 4, upper panel). The band detected at 3070.0 cm −1 could be assigned to alkenyl =C-H stretching or to N-H of a secondary amine. The absorption band observed at 1699.0 cm −1 could be assigned to the carbonyl groups, stretching groups of algal polysaccharides, or to the amide groups of algal proteins. The band at 1464.0 cm −1 could be assigned to the -COO − groups. The detected multiple peaks between 1008.0 and 1190.0 cm −1 , which are characteristic for sugars moieties that could be due to the coupling of the C-O or C-C stretching modes with the C-O-H bending modes. The stretching vibrations of C-O can be triggered from different sources such as carboxylic acid or polyol, where the extracted polysaccharides have a variety of components like neutral sugars, uronic acids, and amino sugars. The band observed at 846.0 cm −1 can be assigned to-C-O-SO 4 of sulfated polysaccharides. Finally, the band at 665.0 cm −1 can be assigned to SO 4 −2 groups. The UV-Visible spectrum of AuNPs after 3 months that prepared by polysaccharides with l-ascorbic acid (AuNPs 3), AuNPs3 were more stable even after more than 3 months comparing with nanogold colloidal solution that prepared by l-ascorbic acid alone as a reducing and stabilizing agent.  (Fig. 4a). This indicates that the presence of OH groups in the polysaccharides is the key group involved in the reduction of Au ions. The absorption band at about 1640.0 cm −1 can be assigned to the stretching vibration of -C = O of associated secondary amide groups 38 . The two observed peaks at nearly 1540.0 and 1450.0 cm −1 can be assigned to the stretching vibration   Fig. 6b).
Safety pattern of the biogenic AuNPs. In vitro viability test was used to investigate the safety patterns of the AuNPs at different concentrations (2.0 to 0.0156 mg/ml). WISH cell line was used as a non-cancerous cell line model to detect the AuNPs safest doses to be used in the proceeding tests. Using MTs assay, the safest AuNPs preparation was found to be AuNPs3 that showed 17.35% maximum toxicity. While, both AuNPs1 and AuNPs2 recorded 18.92% and 51.4% cytotoxic percentages on WISH cells, respectively. All AuNPs recorded more than 90.0% cellular viability (Fig. 7a) at a concentration of 0.031 mg/ml.
In vitro anticancer activities of the biogenic AuNPs. Cytotoxicity assay of the biogenic AuNPs. Anticancer activities of the biogenic AuNPs were studied against A549, CaCo-2, and MCF-7 cancer cell lines upon treatment with AuNPs1, AuNPs2, and AuNPs3 sub IC 50 concentrations. Serious morphological changes in cell structure were observed after treatment. Furthermore, the cytotoxicity results presented in Fig. 7b,c,d indicated that the biogenically-synthesized AuNPs markedly inhibited all tested cancer cell lines with different degrees. The maximum percentages of inhibition on A549 cells after AuNPs1, AuNPs2, and AuNPs3 treatments were 43.6%, 44.5%, and 41.6%, respectively (Fig. 7b), and with IC 50 values of 2.3, 2.3, and 1.4 mg/ml, respectively (Fig. 8b).   8a). It is worth to mention that the recorded IC 50 values of AuNPs1, 2, and 3 on the non-cancerous WISH cell line were 5.08, 0.64, and 5.14 mg/ml, respectively (Fig. 8a). According to these IC 50 values of both cancerous and non-cancerous cell lines, the selectivity index of AuNPs to cancer cells were indicated in Fig. 8b. In general, the maximum selectivity index values recorded after MCF-7 treatment with AuNPs1 and AuNPs3 were 10.37 and 25.5, respectively (Fig. 8b). Furthermore, comparing with the untreated cells (Fig. 9a), after applying the sub IC 50 dose of AuNP1 treatment to MCF-7 (Fig. 9b), AuNPs2 (Fig. 9c), and AuNPs3 (Fig. 9d), apoptotic and dead cells occurred in major parts of the cultured plates with serious changes in cell structure and number.    Transmission electron microscopy (TEM) of AuNPs-treated MCF-7 cell line. As the above mentioned results confirmed that the highest MCF-7 cellular proliferation inhibition percentage was recorded after AuNPs3 treatment. So that, we investigated the internalization of AuNPs2 and AuNPs3 in MCF-7 cell line using TEM imaging technique. Comparing with control (untreated) cells (Fig. 12a), both AuNPs were found in cell vacuoles, cytoplasm (Fig. 12b), and/or in cell perinuclear region (Fig. 12c,d), where AuNPs were taken via endocytosis (Fig. 12d). Also, some changes were recorded in the nucleus and mitochondria morphology. Furthermore, membrane blebbing together with apoptotic bodies (Fig. 12e,f) were also noticed. These results are similar to the results of flowcytometry that confirmed the induction of MCF-7 cell death after AuNPs treatment.

Materials and methods
Microorganisms and cell lines. Microbial   Cultivation of Arthrospira platensis. Arthrospira platensis strain was inoculated into 250.0 ml of modified Zarrouk medium, illuminated with white fluorescent light (2500 Lux) for 16.0 days at 25 ± 2 °C, and the flask was shaken twice per day to keep the culture homogenous. At the end of incubation period, the culture filtrate was collected by centrifugation at 4000 rpm for 30.0 min at 4.0 °C and the recovered filtrate was further purified using a Whatman filter paper to remove any remaining suspended algal biomass 39 .
Biosynthesis of gold nanoparticles. Extraction of water soluble polysaccharides. The total cellular metabolites of Arthrospira platensis were collected from the culture as previously described 39 . Briefly, an aliquot of 500 ml of algal filtrate was boiled for 30 min at 100 °C and allowed to cool down at room temperature. Polysaccharides were precipitated by the addition of fourfold ethyl alcohol 95.0% (v/v). The precipitated polysaccharides were collected by filtration using Whatman filter papers (110 mm) and washed by absolute ethanol and left to dry in oven at 50 °C to complete dryness.
Biosynthesis of AuNPs by thermal reduction method. Sodium tetrachloroaurate (III) dihydrate (NaAuCl 4 ·2H 2 O, Sigma-Aldrich) was used to prepare a stock solution of initial concenration 1.0 mM in ultra-high purified water (Milli-Q plus system, Millipore Co., USA). The prepared solution was boiled for 30.0 min then left to cool down at room temperature. This pre-boiled stock solution was used to prepare AuNPs using two different methods; thermal reduction and l-ascorbic acid reduction methods.
The following experiments were performed using different dilutions of the NaAuCl 4 stock solutions (0.25, 0.50, and 1.00 mM) to adjust the optimum molar ratios used in the biosynthesis of AuNPs. The best NaAuCl 4 concentration was used in further optimization processes to maximize the production of AuNPs as follows.
In  X-ray powdered diffraction (XRD) analysis. The obtained AuNPs preparations were dried out at 50.0 °C for 16.0 h and the purified dried powders were scanned using diffraction angles (2Ɵ) that ranged between 5.0° and 80.0°. The XRD patterns of the produced nanoparticles were measured using a diffractometer (LabX-6100, SHI-MADZU) with a 40-kV voltage and a 30.0 mA electric current employing Cu Kα radiation (λ = 1.5418 Å). The analysis was performed at the Egypt-Japan University of Science and Technology (E-JUST).
Nanoparticles sizes were estimated using XRD measurements by applying Debye Scherrer equation, where strong reflections with large intensities were used to measure the full width at half maximum (FWHM). The Scherrer equation for calculating the particle size is given by: The Scherrer constant (K) in the above formula accounts for the shape of the particle and is generally taken to have the value 0.9 as a good approximation.   where A: the absorbance of the treatment group, A1: the absorbance of the blank, and A0: the absorbance of the control group.
In vitro anticancer activity of green synthesized NPs. Anticancer activities of AuNPs against A549, CaCo-2, and MCF-7 cell lines were investigated using MTS assay protocol as described in the safety assay method. The morphological changes occurred in cancer cells, post AuNPs treatment, were inspected using CKX41 Olympus Inverted Microscope, Japan.
Selectivity index (SI). Cancer cell selectivity index of the biogenic AuNPs was calculated as reported by Koch et al. 42 with a minor modification; where IC 50 nc refers to the IC 50 value of the tested compound on normal cells, IC 50 cc refers to the IC 50 of the tested compound on the tested cancer cell line. Gene expression pattern alternation in MCF-7 cancer cell line after AuNPs treatments. The anticancer molecular mode of action of AuNPs was studied via screening their activities in controlling the expression of Bcl2, IKap-α, and Survivin genes (Table 3) in MCF-7 cells (the most sensitive cancer cell line for AuNPs). After cellular treatment, MCF-7 cell line was cultured in 12.0-well plates (6.0 × 10 3 cell/ml) for 24.0 h along with the resulted non-toxic concentration of AuNPs. After incubation, total cellular RNA was extracted using RNA extraction kit (Qiagen). Then, cDNA was synthesized using Oligo-dT primer and AMV reverse transcriptase (Promega Corp., Madison, WI). β-actin primers were used to amplify the house-keeping, β-actin, gene as an internal control for standardization of PCR products. The RTq-PCR was done using SYBR Green dye (QuantiTect SYBR Green PCR Kits) and Light Cycler fluorimeter Bio-Rad S1000 Tm thermal cycler (Bio-Rad, USA).

Mode of anticancer action of AuNPs treatments against
Transmission electron microscopy for MCF-7 treated cells. The most sensitive cell line to AuNPs treatments was selected to be scanned using Transmission Electron Microscope (JEOL, Japan). The treated MCF-7 cells were collected, fixed, and dehydrated using a series of acetone washes. Then, the cells were passed through a transition solvent such as propylene oxide, infiltrated, and finally embedded in a liquid resin (epoxy and LR Cytotoxicity percentage = (A − A1/A0) × 100 SI = IC 50 nc/IC 50 cc Table 3. Primers sequence used for quantitative real time PCR.

Gene Sequences
Bcl2 F: 5′-TAT AAG CTG TCG CAG AGG GGCTA-3′   R: 5′-GTA CTC AGT CAT CCA CAG GGC GAT -3′   IKap-α  F: 5′-CAT GAA GAG AAG ACA CTG ACC ATG GAAA-3′   R: 5′-TGG ATA GAG GCT AAG TGT AGA CAC G-3′   Survivin  F: 5′-TGC CCC GAC GTT GCC-3′   R: 5′-CAG TTC TTG AAT GTA GAG ATG CGG T- www.nature.com/scientificreports/ White resin). After embedding, the solidified resin block was then sectioned by ultramicrotome, where sections of 50.0-70.0 nm thickness were made using a diamond knife. The sections were mounted on TEM grids and stained with 4.0% Uranyl acetate for 25.0 min. Then, the grids were rinsed four times with pure water before staining with 1.0% lead citrate for 5.0 min and rinsing with pure water. The grids were finally stored in a grid box until examination under TEM.
Statistical analysis. Data are presented as mean ± SD. Two means were compared by Student's t-test and three or more group were meant by one-way analysis of variance (ANOVA) with X tests for pair-wise comparisons. A p value < 0.05 was considered significant. All statistical calculations were conducted using Graph Pad Prism 7 software.

Discussion
Nanoparticles and nanotechnology are playing important roles in different fields such as medicine, biology, physics, chemistry, and sensing due to their distinctive properties 45 . The nanoparticles of noble metals (Cu, Hg, Ag, Pt, and Au) comparing with other metal nanoparticles, are increasingly attracting researchers attention 45 due to their unique optical and electrical properties. For example, AuNPs was used in various applications of interdisciplinary branches of science including medicine, material science, biology, chemistry and physics 46 . The FDA organization has approved the conjugation of anticancer drugs, diagnostic agents, and/or targeting agents to nanobiomaterials to build nanostructure weapons against cancer cells 2 . At the time being, AuNPs are prepared by different green and synthetic techniques with different shapes as nanospheres, nanorods, nanocubes, nanobranches, nanobipyramids, nanoflowers, nanoshells, nanowires, and nanocages 47,48 . Green chemistry is one of the promising research areas in nanotechnology for the fabrication of nanomaterials due to the growing demand to the synthesis of environmentally safe nanomaterials 49 and to reduce the cost and energy consumption associated with the production process using the physical/chemical techniques 50 . The green synthesis approach are nontoxic, one step, easily available in affordable value, and thus are more preferable than the chemical and physical approaches 8 . Metallic nanoparticles have been biosynthesized using various natural resources as algae 39,51 , fungi 52-54 , bacteria 55 , and plants 56,57 as reducing and stabilizing agents for the fabrication of nanoparticles with different morphologies 58 and size distributions. Therefore, in this study, we reported for the first time the green synthesis of biologically effective AuNPs via different concentrations of Arthospira platenisis exopolysaccharides and evaluated their efficacy against various cancer cell lines and microbial strains. The majority of the published research studies reported plant-mediated synthesis processes for the production of AuNPs and the most effective biogenic particles were spherical or nearly spherical shaped AuNPs with diameter size less than 100.0 nm 59 . Our recovered biogenic AuNPs2 showed a range of particle sizes from 8.0 to 40.0 nm, the particles were isotropic, which are well known for small spherical nanoparticles due to their small aspect ratio 60,61 . Each of the prepared AuNPs colloidal solutions expressed a very intense color, which is not found in the case of its parent material as previously reported in the published literatures [62][63][64] . The reason behind these colors is attributed to the collective oscillation of free conduction electrons. The surface plasmon resonance (SPR) absorbance is highly sensitive to the size, nature, temperature, shape of particles, and the environment of the surroundings 65 . Generally, gold nanoparticles show intensive SPR bands in the region of 500.0 to 600.0 nm at the visible spectral region depending on the method of fabrication, the size of the particles, and the surrounding parameters 66,67 . Also, UV-Vis spectrum of the recovered AuNPs showed absorption band at 560.0 nm, which indicate a little red shift as a result of the occurrence of some degree of aggregation. The aggregation states could be detected visually by a change in color of the solution from red to blue or purple. Blue shift was observed in the current study from 550.0 to 530.0, which emphasis the dependence of spectra on the size of AuNPs, as the size of gold nanoparticles decreases, the maximum absorption peaks were shifted to smaller wavelengths, a phenomenon called "Blue Shift" 68 . Different research studies showed that different biological sources which were used for AuNPs biosynthesis significantly affected their biological activities. As the biomolecules extracted from natural sources are used as unique reducing and capping agents for the reduction of metallic ions into differently-shaped and effective nanoparticles, whose specific chemistry are definitely linked to the efficacies of the parent materials 69 . In addition to the type of the capping agents used in NPs preparation, nanoparticles characteristics such as their shape, size, surface chemistry, and charge could affect their pharmacokinetics (absorption, metabolism, distribution, and elimination) 70 . These findings could explain the cytotoxic effects of the prepared AuNPs in the current study, as it was reported that AuNPs with average size < 30.0 mm were more cytotoxic and could be endocytosed by cells 71 . Furthermore, capping agents and NP characteristics such as shape, size, surface chemistry, and charge also influence the safety properties of NPs. Although different research articles confirmed the significant biomedical activities of biogenic metallic nanoparticles, it is very important to detect the hazards associated with the use of biogenic NPs. It is noteworthy that AuNPs safety is a hot topic that has gained great interest due to their potential biotechnological applications, but their safety is still a matter of debate among scientists. A large number of in vitro and in vivo experiments have proved the safety of AuNPs, while others confirmed their toxicity 70 . Different factors such as size, shape, surface capping materials, charges of the surface, dose, and exposure time affect the toxicological properties of the prepared nano-particles 72 . In the current study, our results indicated that the safest concentration of AuNPs on WISH cells was 30.0 μg/ml), which is a very low concentration comparing with both the reported concentrations in the scientific literature and the previously reported concentration of the biologically prepared AgNPs using the same polysaccharides 39 . In addition, another report explained that the biogenic AuNPs exert their toxic effect at concentrations as low as 40.0 μg/ml 73 .
The antibacterial effects of nanomaterials such as silver, gold, copper, titanium, zinc oxide, and magnesium oxide made them potential substitutes or complementary agents for antibacterial therapies 74  www.nature.com/scientificreports/ no clear explanation in the scientific literature on the exact mechanism of NPs antibacterial effects, it is hypothetical that NPs could target the microbial cell membranes and damage the membrane potential. By considering the biomolecules capping of the prepared NPs surface, we believe that their strong antibacterial effects could be related to an easier penetration of the cell membranes by these biomolecules that resulted in increasing toxicity. Our results indicated that Gram negative bacteria were more sensitive to AuNPs treatments and this could be the result of the presence of tough cell wall in Gram-positive bacteria, but gram-negative bacterial cell wall is thinner. Therefore, AuNPs easily penetrate the cell membrane of the gram-negative bacteria and cause damaging effects to the bacterial cell 75 . This finding confirmed the antimicrobial effects of AuNPs that could be used as a potential antimicrobial agent in the future. The different anticancer effects of the biologically synthesized NPs on the same cell lines was found to be related to the nature of the biological capping agents, the metal NPs size distribution and shapes, and 76 the experimental conditions such as pH, temperature, and the concentration of metal salt 8,76,77 . As we mentioned above, the different cytotoxic effects of nanoparticles could be attributed to the different nature of the capping agent present in algal polysaccharides. Although, the variable cytotoxic effects of the biogenic nanoparticles on different mammalian cells could be attributed to the size and level of aggregation of the NPs 72 . Various biomolecular agents were reported to be involved in AuNPs synthesis and stabilization into smaller nanoclusters. These agents could be amino acids, protein side chains, glutathione, phospholipids, and many more agents 72 .
The results of screening the efficacy of the biogenic AuNPs against breast cancer could be summarized in 4 groups 59 , the first group confirmed the cytotoxic effects of biogenic AuNPs against breast cancer cells, while the second one reported no cytotoxic effects were observed upon cancer cells treatment with the nanostructures. In addition, the third group confirmed significant cytotoxic effects of the biogenic AuNPs against breast cancer cells, but low or no cytotoxicity against non-cancerous cells. Meanwhile, the last group didn't record any cytotoxic effects against both cancerous and non-cancerous cells. Interestingly, no reports were found to confirm the higher cytotoxic effects of the biogenic AuNPs on non-cancerous cells over breast cancer cells 59 . In agreement with the third group, the current study confirmed the anticancer selectively effects of the biogenic AuNPs against MCF-7 cell line with low cytotoxic effects against noncancerous cells (about 25 times cytotoxic effects on the MCF-7 cells over the non-cancerous cells). The anticancer effects of the biogenic AuNPs were explained by arresting MCF-7 cells in the S phase and increasing the cellular population in the sub G0 phase. Similar results were found by earlier reports on MCF-7 and S phase arrest in MDA-MB-231 [78][79][80] . Furthermore, decreasing the size of materials to the nanoscale extraordinary increases the reactivity and subsequently the interaction of NPs with the biological entities. Our recovered AuNPs were found to be deposited in the vacuoles, the cytoplasm, and in the perinuclear region of MCF-7 cells. AuNPs may be internalized into these cellular structures and caused different ultrastructural modifications. Generally, positively charged molecules have higher uptake ratio, but poor intracellular stability comparing to neutral or negatively charged molecules 81 . Furthermore, nanoparticles size positively affect their internalization 82 . Two main AuNPs internalization mechanisms were reported: membrane-bound vesicles and endosomes 83 . It has been indicated that AuNPs rods may be endosomes internalized by vesicular bodies into human dermal fibroblasts and colon adenocarcinoma 84,85 . Other studies reported that AuNPs could be phagocytic internalized in A549 and HBL-100 cells 86,87 . Furthermore, AuNPs can be located in the cytosol, lysosomes, and perinuclear region as aggregates or singletons 86,87 . The abilities of biogenic nanoparticles to alter the expression patterns of cancer cell genes and their genotoxic effects in different in vitro and in vivo models were reported 51,88 . Furthermore, the abilities of AuNPs to induce cellular apoptosis by decreasing the expression levels of Survivin and Bcl-2 were confirmed. Choudhury et al., reported a reduction in the level of Bcl-2 protein (anti-apoptotic protein) in A549 cells after treatment with 40 nm AuNPs 89 . Also, Selim et al., indicated that AuNPs could decreased the level of Bcl-2 proteins in the treated MCF-7 cells 90 .

Conclusion
In the present work, it has been demonstrated that Arthospira platensis exopolysaccharides (EPS) are capable of reducing gold ions into three different AuNPs and the generated nanoparticles were stable for more than 3 months. The biosynthesized nanoparticles showed potent antimicrobial and cytotoxic effects against the tested cell lines and microbial strains. This study opens the door for the usage of the biogenic AuNPs alone or in parallel with chemotherapies and antibiotics for future cancer and microbial therapies, respectively.

Future prospective
Although we provided strong and comprehensive in vitro results, further biological applications and in vivo studies are required to confirm the reliability and efficacy of these AuNPs in animal models, which is our future prospective.

Data availability
All data generated or analyzed during this study are included in this published article [and its supplementary information files].